title: The CP2 domain of leucyl-tRNA synthetase is crucial for amino acid activation and post-transfer editing.
abstract: Leucyl-tRNA synthetase (LeuRS) has an insertion domain, called connective peptide 2 (CP2), either directly preceding or following the editing domain (CP1 domain), depending on the species. The global structures of the CP2 domains from all LeuRSs are similar. Although the CP1 domain has been extensively explored to be responsible for hydrolysis of mischarged tRNALeu, the role of the CP2 domain remains undefined. In the present work, deletion of the CP2 domain of Giardia lamblia LeuRS (GlLeuRS) showed that the CP2 domain is indispensable for amino acid activation and post-transfer editing and that it contributes to LeuRS-tRNALeu binding affinity. In addition, its functions are conserved in both eukaryotic/archaeal and prokaryotic LeuRSs from G. lamblia, Pyrococcus horikoshii (PhLeuRS), and Escherichia coli (EcLeuRS). Alanine scanning and site-directed mutagenesis assays of the CP2 domain identified several residues that are crucial for its various functions. Data from the chimeric mutants, which replaced the CP2 domain of GlLeuRS with either PhLeuRS or EcLeuRS, showed that the CP2 domain of PhLeuRS but not that of EcLeuRS can partially restore amino acid activation and post-transfer editing functions, suggesting that the functions of the CP2 domain are dependent on its location in the primary sequence of LeuRS.
authors.affiliation: State Key Laboratory of Molecular Biology and Graduate School of the Chinese Academy of Sciences, Institute of Biochemistry and Cell Biology, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
authors.forename: Xiao-Long
authors.initials: XL
authors.lastname: Zhou
authors.forename: Bin
authors.initials: B
authors.lastname: Zhu
authors.forename: En-Duo
authors.initials: ED
authors.lastname: Wang
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D004926:Escherichia coli / Q000378:metabolism*; D016829:Giardia lamblia / Q000378:metabolism*; D006868:Hydrolysis; D007930:Leucine / Q000737:chemistry; D007935:Leucine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D043902:Pyrococcus horikoshii / Q000378:metabolism*; D017393:RNA Editing; D012343:RNA, Transfer / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
chemical_list: D012343:RNA, Transfer; D007935:Leucine-tRNA Ligase; D007930:Leucine; D000409:Alanine